High throughput screening model for hepatitis C virus (HCV) resistant drugs and application thereof

An anti-hepatitis C, high-throughput technology, applied in genetic engineering, cell biology, and medical fields, can solve problems such as unsatisfactory, human factors and large errors, difficult to achieve high-throughput or automated applications, etc.

Active Publication Date: 2014-06-25
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the biggest drawback is that it is difficult to realize automatic monitoring, especially it cannot be sorted by FACS, so it is difficult to apply to high-throughput screening
[0009] The above methods may have complicated operation process or read information, or have low response efficiency, and often cannot meet the needs of scientific research or even drug screening
In comparison, the method developed by the Charles Rice group is relatively simple to operate and easy to observe, but it relies on the pattern change of the intracellular fluorescent protein (from point-like aggregation to diffusion), which has large human factors and errors, and is difficult to achieve High-throughput or automated applications

Method used

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  • High throughput screening model for hepatitis C virus (HCV) resistant drugs and application thereof
  • High throughput screening model for hepatitis C virus (HCV) resistant drugs and application thereof
  • High throughput screening model for hepatitis C virus (HCV) resistant drugs and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Anti-HCV drug high-throughput screening model and construction method thereof

[0080] 1. Construction of reporter system plasmid

[0081] Using the plasmid pLentiCMVMCSSBsd as the backbone, the plasmid map is as follows Figure 12 As shown, the full sequence of the plasmid is shown in Seq ID No.9.

[0082] Cloning is a two-step process:

[0083] Step 1: Delta-tk, 2A, and mCherry reporter gene modules correspond to primers 1-6 in Table 1, respectively, and are amplified by PCR. The gene templates are stored by Wei Wensheng Laboratory, School of Life Sciences, Peking University. Then the corresponding restriction endonucleases were used to carry out enzyme digestion, and sequentially loaded into the multiple cloning site behind the tight-TRE promoter of the pEN_TTmcs plasmid. Thus, the complete sequence from tight-TRE to mCherry (including stop codon) was obtained.

[0084] Then, the CMV promoter that comes with the pLentiCMVMCSSBsd plasmid was excised with...

Embodiment 2

[0097] Example 2 NS3 / 4A specific inhibitor VX-950 can significantly inhibit the fluorescent signal of the system

[0098] This example was developed based on the physiological activity of HCV NS3 / 4A protease. In order to verify the rigor and sensitivity of the reporter system constructed in Example 1, and whether it can be applied to compound screening, the following experiments were designed:

[0099] The HCV host cell Huh7.5 containing the reporter system was mixed with 10 per well 5 The cells were spread evenly on a 12-well plate. Cultured in DMEM medium at 37°C in an incubator with 5% carbon dioxide concentration.

[0100] The DMEM medium is DMEM (Life Technologies), 10% FBS (Thermo Scientific), 1×NEAA (Life Technologies) and contains 1× double antibody (penicillin-streptomycin).

[0101] The next day, use doxycycline (2 μg / ml) and HCVcc (3.6×10 4 FFU / ml) of DMEM to renew the medium in each well.

[0102] At the same time, with DMSO as the control group, different volu...

Embodiment 3

[0104] Example 3 Using Flow Cytometry (FACS) to Realize High Throughput Analysis

[0105] In order to further verify the sensitivity of the reporter system of the present invention to HCVcc infection, and whether FACS can be successfully used for high-throughput analysis, the following experiments were designed:

[0106] 1. Prepare cells and set up analysis samples

[0107] 2×10 per well 5 Density of cells The monoclonal Huh7.5 cells with the best performance selected in Example 1 were spread in turn on a 6-well plate, with a total of 14 wells, including a control group and 13 experimental groups.

[0108] On the next day, referring to the method of using the fluorescent signal in Example 2, the medium was renewed with DMEM containing doxycycline, 2 ml per well. Add 0.6ml of HCVcc virus stock solution and 0.4ml of DMEM medium (and supplement 1μl of doxycycline stock solution with a concentration of 2mg / ml) at the highest concentration (well 14), a total of 3ml, and the calcu...

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Abstract

The invention provides a novel high throughput screening model for hepatitis C virus (HCV) resistant drugs. The high throughput screening model can be applied to research and drug screening of human HCV infection mechanisms, and is a reporting system carrying out efficient response aiming at HCV infection. After the HCV invades cells, protease NS3/4A expressed by the HCV is about to cut and release a tetracycline-regulated transactivator (rtTA) anchored on a mitochondrial outer membrane by an MAVS (mitochondrial antiviral signaling) protein, expression of a reporter gene is activated in the presence of doxycycline or tetracycline, and obvious fluorescence signals are observed in a certain time; or cell death is caused in the presence of GCV (gancilovir). The system is simple to operate, has short experimental period, high efficiency and strong stability, can be widely applied to real-time quantitative observation of HCV infection, flow cell sorting, high throughput compound screening, high throughput host cell target gene screening and molecular mechanism research of HCV infection.

Description

technical field [0001] The invention relates to the fields of medicine, genetic engineering and cell biology, in particular to a high-throughput screening model for anti-hepatitis C virus drugs and its application. Background technique [0002] HCV (Hepatitis C virus) is a hepatitis virus that was not reported for the first time until 1989 (Choo et al., 1989). It is a single-stranded RNA virus that causes hepatitis C and is one of the main causes of human viral hepatitis. one. As of 2005, about 3% of the world's population, more than 170 million people, were infected with hepatitis C virus. HCV is a virus that does not cause cell pathological changes in the liver. It is a member of the Flaviviridae family. Acute hepatitis may occur after infection, and acute hepatitis can be cured, while about 70% will develop into chronic hepatitis and eventually cause liver disease. Serious diseases such as cirrhosis and liver cancer (Hoofnagle et al., 2002). So far, no anti-HCV vaccine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12Q1/04
Inventor 魏文胜任庆鹏李婵
Owner PEKING UNIV
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