Drug screening model of targeting coronavirus protease and application thereof

A coronavirus and protease technology, applied in the direction of microorganisms, microorganism-based methods, microorganism measurement/inspection, etc., can solve the problems of high risk for experimenters, virus leakage, inconvenient operation, etc., to avoid virus infection and dynamic screening Effect

Active Publication Date: 2011-04-20
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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Problems solved by technology

For virus-infected cell models, BSL3 laboratories must be used for highly pathogenic viruses such as SA

Method used

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  • Drug screening model of targeting coronavirus protease and application thereof
  • Drug screening model of targeting coronavirus protease and application thereof
  • Drug screening model of targeting coronavirus protease and application thereof

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Embodiment Construction

[0019] 1. Drug screening model targeting coronavirus PLP protease: clone the PLP2-TM fragment of NL63 into the vector pcDNA3.1-V5 / His B (Xho I and Apa I). The amino acid sequence of the nsp2 / 3 site recognized by PLP2 -FTKLAG↓GK- is carried on the N segment of PLP2. Using SEAP-basic vector (Clontech) as a template, PCR obtained the SEAP fragment (with EcoR I and Kozak sequences and SEAP ATG at the 5' end, and XhoI at the 3' end), and cloned it into pcDNA-V5 / HisB PLP2-TM At the 5' end of the fragment, a 5'-SEAP-PLP2-TM-3' DNA construct (pcDNA-SEAP-PLP2-TM) was obtained. The recombinant DNA plasmid was transfected into Hela cells, and 50 μl of supernatant was collected at each time point of 12h, 24h, 36h, 48h and 60h after transfection. The SEAP activity in the supernatant was detected by Chemiluminescent Assay (the reagent was Great EscAPe SEAP Chemiluminescent and Fluorescence Detection Kits, Clontech Company).

[0020] In order to confirm that PLP2 can release SEAP into the ...

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Abstract

The present invention provides a novel cell-based drug screening model of targeting coronavirus protease. In the processing products of the coronavirus polyprotein 1a(1ab), nsp3, nsp4 and nsp6 contain transmembrane (TM) domains which can mediate the protein to locate on the cell endoplasmic reticulum (ER) membrane. Using the characteristics that nsp3, nsp4 and nsp6 contain transmembrane domains and the protein is located in the cell, the present invention constructs a eukaryotic expression DNA construct by secretion reporter gene, wherein the eukaryotic expression DNA construct consists of coronavirus proteases - protease recognition sites - secretion reporter genes. After cell transfection, the expression products of the active releasing reporter gene are cut by the coronavirus protease and are secreted into the cellular supernatant; the activity of the coronavirus protease is detected by the activity of the reporter genes in the cellular supernatant and is used for coronavirus drug screening.

Description

Technical field: [0001] The invention relates to a bioengineering detection product and its application. Background technique: [0002] Coronavirus is one of the main viruses that cause respiratory infections. At present, there are five kinds of coronaviruses that cause human respiratory tract infection, 229E, OC43, SARS-CoV, NL63 and HKU1. About 30% of human respiratory infections are caused by two coronaviruses, 229E and OC43; SARS-CoV is a highly pathogenic respiratory pathogen that newly appeared in early 2003 and caused human acute respiratory syndrome (SARS), and the infection mortality rate is about 9 %, is currently the most infectious and pathogenic new human coronavirus. In the following three years, two new human coronaviruses NL63 (2004) and HKU1 (2005) were successively discovered from patients with respiratory tract infection. It can be predicted that many unexplained respiratory infections may be caused by other undiscovered human novel coronaviruses. [0...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/37C12R1/93
Inventor 陈忠斌陈晓娟邢雅玲杨宇东
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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