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High-throughput screening method and application of drug for inhibiting human adenovirus proliferation

A human adenovirus, screening method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, viruses, etc., can solve the problems of heavy workload, high cost, and long time consumption

Pending Publication Date: 2021-04-02
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to establish a high-throughput screening method for measuring the inhibition of adenovirus proliferation by drugs, so as to solve the problem of heavy workload, long time consumption and high cost when using existing conventional methods to detect the inhibitory effect of drugs on adenoviruses. It is helpful for the screening of new anti-adenoviral drugs and provides an effective screening platform for the research and development of new anti-adenoviral drugs

Method used

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  • High-throughput screening method and application of drug for inhibiting human adenovirus proliferation
  • High-throughput screening method and application of drug for inhibiting human adenovirus proliferation
  • High-throughput screening method and application of drug for inhibiting human adenovirus proliferation

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Embodiment 1

[0059] Example 1 Establishment of a high-throughput screening method for determining drugs that inhibit the proliferation of human adenoviruses

[0060] 1. Preparation of GFP-integrated human adenovirus

[0061] Use pAdTrack-CMV (https: / / www.addgene.org / 16405 / ) and pAdEasy-1 (https: / / www.addgene.org / 16400 / ) plasmids to construct plasmid pAd-YFP (SEQ ID NO.1), Transfect the plasmid pAd-YFP into 293FT cells to obtain GFP-integrated human adenovirus. For details, refer to https: / / media.addgene.org / data / 49 / 60 / 162ad396-af64-11e0-90fe-003048dd6500.pdf. In simple terms, the method is:

[0062] 1) The plasmid pAdTrack-CMV was linearized with PmeI restriction endonuclease.

[0063] 2) Co-electrotransfer the linearized pAdTrack-CMV and adenovirus backbone vector pAdEasy-1 plasmids into Escherichia coli BJ5183 cells. Kanamycin was used for monoclonal screening, and the obtained plasmids were identified by enzyme digestion, and the correctly identified plasmids were transformed into DH...

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Abstract

The invention discloses a high-throughput screening method and application of a drug for inhibiting human adenovirus proliferation. A human adenovirus marked with GFP in the patent can express green fluorescent protein after infecting with HEK-293 cells, so that the infected cells are green under a fluorescence microscope. The anti-human adenovirus drug activity is realized through the following steps: detecting the change of the adenovirus content in the supernatant of a culture medium after the anti-adenovirus drug and the human adenovirus are added into the HEK-293 cells by using flow cytometry. The specific operation comprises the following steps: taking a proper amount of the virus supernatant, adding the virus supernatant into a certain quantity of the HEK-293 cells again, and determining the ratio (cells with green fluorescence) of the adenovirus infected cells in the cells by flow cytometry so as to evaluate whether the added drug has an obvious inhibition effect on adenovirusproliferation or not. According to the method, whether candidate drug molecules have the effect of inhibiting human adenoviruses or not can be screened quickly, simply, conveniently and effectively invitro in a low-cost and high-throughput manner. According to the method, the research and development of novel anti-human adenovirus drugs can be accelerated.

Description

technical field [0001] The invention relates to a high-throughput screening method for drugs inhibiting the proliferation of human adenoviruses and the application thereof, which can quickly, simply, effectively and high-throughput screen whether the drugs have the effect of inhibiting the proliferation of human adenoviruses in vitro, and contribute to Development of new anti-adenoviral drugs. Background technique [0002] Human adenoviruses are linear, double-stranded, and icosahedral DNA viruses. So far, 70 human adenoviruses have been identified, which are divided into 7 types (A-G) according to their genetic relationship. Different adenovirus species have specific organ preferences, C, E, and B viruses infect the respiratory tract; some B viruses infect the urinary tract; A and F target organs of infection are the gastrointestinal tract; D mainly infects the eyes, which may be related to Different adenoviruses have different receptors. C (serology 1, 2, 5) categories m...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12N15/85C12N15/65C12N7/01C12R1/93
CPCC12Q1/025C12N15/85C12N15/65C12N7/00G01N2333/075G01N2800/107C12N2710/10021C12N2710/10052Y02A50/30
Inventor 谷峰商璐
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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