Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

C genotype entecavir-resistant mutation HBV stable replication and expression cell line

A cell line and C gene technology, applied in genetic engineering, plant genetic improvement, cells modified by introducing foreign genetic material, etc., can solve the problem of affecting virus phenotype, not fully reflecting the phenotypic characteristics of drug-resistant strains, and drug resistance rate Increase and other issues

Inactive Publication Date: 2011-06-22
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
View PDF1 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the above drug-resistant mutant strains are artificially produced by site-directed mutagenesis techniques, and only non-naturally occurring mutant strains are obtained. If the virus source of the cell model is not directly isolated from patient serum, this non-naturally occurring mutant strain Mutations can affect the phenotype of the entire virus
Normally, when HBV produces drug-resistant mutations, it will produce compensatory mutations to restore its own replication ability. Artificial mutations will modify some natural genetic characteristics of the virus, which cannot fully reflect the phenotypic characteristics of drug-resist

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • C genotype entecavir-resistant mutation HBV stable replication and expression cell line
  • C genotype entecavir-resistant mutation HBV stable replication and expression cell line
  • C genotype entecavir-resistant mutation HBV stable replication and expression cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of 1.1 times longer HBV expression vector

[0041] (1) Preparation of full-length circular HBV template The full-length HBV genome (S1145) cloned in the pGEM-Teasy vector was digested with BspQI / ScaI, purified and recovered a 3.2kb fragment, and 10 μL of the digested product was ligated with T4 DNA Enzyme ligation was performed overnight, and the ligation product was used for later use.

[0042] (2) Amplification of double HBV fragments Referring to the S1145 sequencing results, two pairs of primers for amplifying HBV were designed and synthesized according to the literature [Jiang Linbin, et al. PLA Medical Journal, 2010, 35:635-638] (Table 1). Take 5 μl of the circularized HBV DNA product as a PCR template, use the A / B primer pair to amplify a 2735bp HBV partial genome fragment, and use the C / D primer pair to amplify a 643bp HBV partial genome fragment, respectively clone the AB and CD fragments in pGEM-Teasy vector.

[0043] Table 1 The seque...

Embodiment 2

[0046] Example 2 Stable cell line screening

[0047] (1) Plasmid DNA transfection and cell clone screening Extract the transfection-grade recombinant plasmid pTriEx-1.1-HBV, and quantify the concentration with an ultraviolet spectrophotometer. HepG2 cells were cultured at 37°C in complete DMEM medium containing 5% CO2 and 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. 6h 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 continue screen...

Embodiment 3

[0049] Example 3 Detection of the constructed cell lines of the present invention:

[0050] 1. Expression of major HBV antigens

[0051] (1) ELISA detection Collect HepG2.A64 cell line 5th, 10th, 15th, 20th, 25th, 30th, 35th, 40th, 45th, 50th, 55th, 60th generation cell culture supernatant, ELISA (double antibody sandwich method) detection For the expression of HBsAg and HBeAg, the wavelength was set at 450 nm. With the increase of cell passage number, the expression of antigen gradually increased and tended to be stable, the average OD value of HBsAg: 0.67±0.42, HBeAg: 2.03±0.61( figure 2 ).

[0052] (2) Immunohistochemistry Put polylysine-treated slides in a 6-well plate, add HepG2.A64 cells 5×10 5 / well, each 2 wells allowed the cells to slide for 72 hours, and after the cells were fixed, the two-step method was used for immunohistochemical staining. The primary antibody was rabbit anti-HBs / anti-HBc, diluted at 1:100; To amplify the binding signal, the secondary antibo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention constructs a hepatitis B virus stable replication and expression cell line. The virus comes from a C genotype HBV strain epidemic in China, and the DNA polymerase/reverse transcriptase area of the cell line contains nucleoside analogue entecavir-resistant mutational sites. By a screening method, a virus genome is integrated onto a cell chromosome of a human hepatocarcinoma cell line HepG2, and autonomous replication and complete life cycle of the virus are generated. The cell line is stable in antigen secretion, active in virus replication and stable in technical parameters, can provide stable and stable cell models for evaluating HBV-resistant medicaments and developing new medicaments, and provides guidance for clinical treatment.

Description

Technical field: [0001] The invention relates to a stable replication and expression cell line of hepatitis B virus (HBV), especially the contained virus is derived from my country's C genotype HBV strain, and its DNA polymerase / reverse transcriptase region contains the nucleoside analog entecavir (ETV ) The virus stably replicates and expresses the drug-resistant mutation site in a cell line. Background technique: [0002] Establishing a stable replicating cell model of a certain virus, producing a complete virus life cycle and capable of long-term continuous secretion of virus particles, is an essential tool for in vitro antiviral drug screening and research on antiviral mechanisms. Especially in experiments that require standardization, such as antiviral drug sensitivity experiments, the establishment and application of stable replication virus expression cell lines are particularly important. [0003] Since the different reading frames and regulatory sequences of the hep...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N5/10C12N15/51C12Q1/02
Inventor 刘妍王琳徐东平刘文思兰兰戴久增许智慧
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com