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GPCR-targeted drug screening system as well as construction and application thereof

A drug and targeting technology, applied in biochemical equipment and methods, microbial assay/inspection, introduction of foreign genetic material using vectors, etc. Cytotoxicity, Tango system instability and other problems, to achieve the effect of saving experimental operations and costs, saving experimental costs, and simple detection steps

Inactive Publication Date: 2018-11-06
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Due to the shortcomings of the existing Tango system, such as instability, high cost, and cumbersome operation; and its final detection system is not based on living cells, it cannot directly reflect the cytotoxicity of the ligand to be tested; obviously, this makes it unable to be used as a long-term and widely used screening system

Method used

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  • GPCR-targeted drug screening system as well as construction and application thereof
  • GPCR-targeted drug screening system as well as construction and application thereof
  • GPCR-targeted drug screening system as well as construction and application thereof

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Embodiment

[0062] Such as figure 1 As shown, a GPCR-targeted drug screening system Piggybac-Tango, including using Piggybac as a carrier, connecting β-arrestin and tobacco etch virus protease (TEV protease) to form PB-β-arrestin-TEVNia; using Piggybac as a carrier, The Tet response element and GFP were connected to form PB-TRE-GFP.

[0063] The construction method of the above-mentioned GPCR-targeted drug screening system Piggybac-Tango is as follows:

[0064] 1. Construction of the Piggybac-Tango system plasmid:

[0065](1) Construct the PB-β-arrestin-TEV Nia plasmid with Puromycin selection marker, the strategy is to use AC133-CAR piggyBac transposon vector (SEQ ID NO: 1) as the backbone, and pass XbaI (NEB, R0145S) and EcoRI (NEB, R3101L) double digestion to obtain linearized PB-T2A-Puro vector. Adopt the method of bridging PCR, design bridging primer, take F1, R1 as primer (table 1), take the cDNA of 293T cell as template, use Max Super-Fidelity DNA Polymerase (Vazyme, P505-d3) ...

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Abstract

The invention provides a GPCR-targeted drug screening system as well as building and application thereof. The GPCR-targeted drug screening system is characterized by comprising PB-beta-arrestin-TEV Nia formed by connecting beta-arrestin with tobacco etch virus protease and PB-TRE-GFP formed by connecting a Tet response element with GFP. Compared with the previous drug screening system, the GPCR-targeted drug screening system PiggyBac-Tango has the advantages of high stability (the composition element is inserted into the genome by using PiggyBac, and long-term stable expression can be achieved), high sensitivity (the exogenous gene integration form is multicopy, and the detection signals are amplified), high signal-to-noise ratio, low cost (no detection reagent is needed), and in-vivo detection (the cytotoxicity of the ligand to be detected can be reflected). The PiggyBac-Tango system is used as an effective tool for researching and developing GPCR-targeted drugs.

Description

technical field [0001] The invention relates to a GPCR-targeted drug screening system and its construction and application. Background technique [0002] The G protein-coupled receptors (GPCRs) family is the largest membrane protein family in humans, and it is also one of the most successful drug targets in current research. So far, about 34% of the marketed drugs have targeted GPCRs point 1-3 . According to statistics, from 2011 to 2015, sales of drugs targeting GPCRs accounted for 27% of the global market, with sales reaching 890 billion US dollars 2，4 . Although GPCRs account for the largest proportion of all druggable targets, three-quarters of GPCR members have not yet been developed into drug targets; only about 80 GPCR molecules currently have corresponding small molecule drugs 5，6 . Therefore, there is an urgent need for research and development of GPCR ligands based on major diseases. One of the challenges in GPCR-targeted drug screening is the construction of...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/65C12Q1/02
CPCG01N33/5008C12N9/506C12N15/65
Inventor 张玉黄行许黄诚
Owner SHANGHAI TECH UNIV