Luciferase complementary system for high-throughput screening of new crown drugs as well as construction method and application of luciferase complementary system

Abstract

The invention discloses a luciferase complementary system for high-throughput screening of new crown drugs as well as a construction method and application of the luciferase complementary system, and belongs to the technical field of drug screening. The system comprises a cell strain for expressing and researching an exogenous gene, an expression vector containing 3CLPRO and an expression vector connected with a firefly luciferase fragment, the construction method of the system comprises the following steps: respectively inserting the C end and the N end of firefly luciferase into the upstream and downstream of a cutting element of 3CLPRO to construct fluorescent complementary plasmids; cells are split by using a cell lysis solution to expose fluorescein, and then the intensity of firefly fluorescein is detected by using a microplate reader by using the characteristic of bioluminescence reaction generated by the fluorescein and a luciferase detection buffer solution, so that the inhibition condition of the drug on the new coronavirus replication protein 3CLPRO protein is presented. The method has the advantages of high detection sensitivity, strong specificity and low cost, and can be applied to screening of new crown drugs.

Description

technical field [0001] The invention belongs to the technical field of drug screening, and in particular relates to a luciferase complementary system for high-throughput screening of new crown drugs, and a construction method and application thereof. Background technique [0002] 3CL PRO It is the protease of the new coronavirus and a key protein for virus replication. The latest research report shows that the organic selenium compound Ebselen (a new FDA-approved anti-inflammatory drug, can effectively inhibit the replication of HIV-1 capsid protein, can penetrate the blood-brain barrier, and has anti-inflammatory, antioxidant, anti-tumor and anti-viral activities) Mainprotease (M), a key drug target for novel coronavirus pneumonia pro ; aka 3CL PRO ) protease has a strong inhibitory effect, and studies have shown that Ebselen can inhibit 3Cl at the cellular level PRO Therefore, the drug can be used as a positive control drug for the successful establishment of the detec...

AI Technical Summary

Problems solved by technology

[0003] At present, the existing drug screening methods are divided into virtual drug screening and high-throughput screening. Although virtual drug screening has the advantage of rapid screening, this technology can only predict the possible activity of the compound and lacks biological activity testing. The effectiveness of the screened drugs cannot be fully guaranteed; while high-throughput screening includes biochemical screening systems using purified target proteins and affinity-based screening systems

When considering protease inhibitors as targets, the most important thing is to detect protease activity, and this traditional high-throughput screening method often involves protein purification and in vitro experiments for enzyme activity inhibition, which is inefficient and requires expensive equipment , cannot satisfy the high-throughput screening in vivo, so further improvement of the existing high-throughput drug screening method system is needed

[0004] At present, although the therapeutic drugs against the new coronavirus have been reported, with the continuous mutation of the new coronavirus, there is still a long way to go to develop and screen the therapeutic drugs for the new coronavirus

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