Method for screening antiviral medicament for degrading BST-2 activity by antagonistic Vpu

A BST-2, antiviral drug technology, applied in the field of medicine, can solve the problems of rapid virus mutation and drug resistance, and achieve the effects of improving accuracy, avoiding false positives, and improving drugability

Inactive Publication Date: 2010-08-04
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogenic virus is naturally highly mutated, and under the selection pressure of drugs, it is easy to cause rapid mutation of the virus and thus drug resistance.

Method used

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  • Method for screening antiviral medicament for degrading BST-2 activity by antagonistic Vpu

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Construction of the monoclonal Hela cell line stably expressing Vpu

[0020] (1) Construction of Vpu expression vector

[0021] The vpu gene fragment in the BH10 plasmid (NIH AIDS Research&Reference Reagent Program) was amplified by PCR. Primers used: upstream primer 5′-TCT GCA GAA TTC GGG AAA GAC GCA AG-3' (as shown in SEQ ID NO.1), downstream primer 5'AAG CCG CTC GAG CCA TAA TAG ACT GTG AC-3' (shown in SEQ ID NO.2).

[0022] reaction system:

[0023]

[0024] Reaction conditions: 94°C for 45s, 68°C for 60s, a total of 30 cycles.

[0025] The amplified vpu fragment was inserted into the multiple cloning site (EcoRI and XhoI) of the eukaryotic cell expression vector pcDNA3.1(+) (Invitrogen) to obtain the plasmid pcDNA3.1-vpu expressing the HIV-1 accessory protein Vpu.

[0026] (2) Establishment of a monoclonal Hela cell line stably expressing Vpu

[0027] Hela cells were seeded in 6-well plate 2×10 5 , 37°C, 5% CO 2 After culturing for 24 hou...

Embodiment 2

[0029] Embodiment 2: the screening process of medicine

[0030] (1) Cell culture

[0031] The Hela-Vpu cells were cultured. After the cells filled the culture flask, the old medium was discarded and digested with a digestive solution containing 0.25% trypsin and 0.02% EDTA. When the cells become round, discard the digestion solution, immediately add high-sugar DMEM medium (HyClone) containing 10% FBS (fetal bovine serum), and gently blow the bottom of the bottle with a pipette to completely separate the cells from the bottom of the bottle and disperse them into single cells. cell suspension. After counting on a hemocytometer, 1 × 10 cells were seeded in each well of a 96-well cell culture plate.4 After culturing for 24 hours, add drugs for treatment.

[0032] (2) Sample preparation

[0033] Compound sample: Dissolve 10 mg of pure compound in 1 ml of DMSO, dilute to 1 mg / ml with 50% DMSO, take 1.0 μl to act on 100 μl of cell system, so that the final concentration is 10 μg / m...

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Abstract

The invention discloses a method for screening an antiviral medicament for degrading BST-2 activity by antagonistic Vpu, which comprises the following steps: adopting a monoclonal Hela cell line which can stably express the Vpu protein and coating the monoclonal Hela cell line into a cell culture plate; incubating a BST-2 antibody and corresponding horse radish peroxidase HRP labeled secondary antibody and coating in cells of the cell culture plate; and finally, developing color through a TMB substrate, and determining BST-2 antigenic content on the cell surface according to an OD value at 450nm. The method for screening the antiviral medicament for degrading BST-2 activity by antagonistic Vpu can quickly and effectively screen the antiviral activity of the medicament to be tested with high flux, and is an achievable method for screening safe, reliable and effective anti-HIV virus medicaments.

Description

technical field [0001] The invention relates to the field of drugs, in particular to a screening model for anti-AIDS drugs, that is, a method for screening antiviral drugs that antagonize the activity of Vpu degrading BST-2. Background technique [0002] AIDS (acquired immunodeficiency syndrome, AIDS) is caused by human immunodeficiency virus (human immunodeficiency virus, HIV) infection leading to human immunodeficiency, and prone to opportunistic infection and tumor clinical syndrome. In the past 20 years, more than 20 million people have died, and the number of HIV-infected people in the world has reached about 40 million. AIDS has not only become a serious health problem in my country, but also causes hundreds of billions of direct economic losses every year. According to the Ministry of Health of China, by the end of 2009, there will be about 740,000 HIV-infected people in China; in 2009, the number of new HIV-infected people reached 48,000. [0003] Although the trad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531G01N21/31
Inventor 岑山张全李晓宇魏晓露刘振龙贾平平周金明
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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