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Novel horizontal screening system construct by recipient cell G protein coupling and applications

A technology of coupled receptors at the cellular level, applied in the determination/inspection of cells and microorganisms modified by the introduction of foreign genetic material, and the introduction of foreign genetic material using vectors, etc., can solve the overall signal inactivation, hinder G protein binding, etc. question

Inactive Publication Date: 2008-12-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Arrestin family molecules bind to the 7TM receptor and sterically hinder G protein binding, leading to overall signaling inactivation

Method used

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  • Novel horizontal screening system construct by recipient cell G protein coupling and applications
  • Novel horizontal screening system construct by recipient cell G protein coupling and applications
  • Novel horizontal screening system construct by recipient cell G protein coupling and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] (1) Cloning the HM74a gene from human placenta genomic DNA, cloning the β-arrestin2 gene from HEK293 total RNA by RT-PCR, and cloning the N-terminus of the DnaE gene (DnaE-N ) and C-terminal (DnaE-C); construction of plasmid i: pCMV-flag-HM74a and plasmid ii: pCDNA-β-arrestin2.

[0083] (2) Anacystis nidulans R2DnaE-C (1-36aa) and two Renilla luciferase gene C-terminals (rLuc-C, 111-311aa) were spliced ​​together by overlap PCR to splice DnaE-C and rLuc-C, and in DnaE 5 amino acid sequences of CFNGT were added between -C and rLuc-C, and a GC linker (whose amino acid sequence was GGGGSG) was connected to the N-terminal of DnaE-C; , 1-110aa) and Anacystis nidulans R2DnaE-N (37-152aa) were spliced ​​together by overlapping PCR to rLuc-N and DnaE-N, and two amino acid sequences of GS were added between rLuc-N and DnaE-N, and rLuc- The N-terminal of N is also connected to a GC linker.

[0084] (3) Insert the above-mentioned constructed DnaE-C-rLuc-C and rLuc-N-DnaE-N into ...

Embodiment 2

[0091] (1) Cloning the HM74a gene from human placenta genomic DNA, cloning the β-arrestin2 gene from HEK293 total RNA by RT-PCR, and cloning the N-terminus of the DnaE gene (DnaE-N , 37-141aa) and C-terminal (DnaE-C, 1~36aa);

[0092] (2) Nostoc punctiforme DnaE-C (1-36aa) and two Renilla luciferase gene C-terminals (rLuc-C, 229-311aa) were spliced ​​together by overlap PCR to splice DnaE-C and rLuc-C, and in DnaE 5 amino acid sequences of CFNGT were added between -C and rLuc-C, and a GC linker (whose amino acid sequence was GGGGSG) was connected to the N-terminal of DnaE-C; the N-terminals of two Renilla luciferase genes (rLuc-N , 1-229aa) and Nostoc punctiforme DnaE-N (37-141aa) were spliced ​​together by overlapping PCR to rLuc-N and DnaE-N, and two amino acid sequences of GS were added between rLuc-N and DnaE-N, and rLuc- The N-terminal of N is also connected to a GC linker.

[0093] (3) Insert the above constructed DnaE-C-rLuc-C and rLuc-N-DnaE-N into plasmid i and plas...

Embodiment 3

[0096] Example 3: Detection of endocytic activity of human cannabinoid receptor CB1

[0097] According to the method of Example 1, the plasmid vectors pCMV-Flag-CB1-rLucN-DnaEN and pcDNA-βArrestin2-DnaEC-rLucC were constructed, and then pCMV-Flag-CB1-rLucN-DnaEN and pcDNA-βArrestin2- The DnaEC-rLucC expression vector was co-transfected into HEK293 cells, and the stable expression cell lines were obtained by G418 (800 μg / ml) selection for 2-3 weeks. Stable expressing cells were digested with trypsin-EDTA, transferred to 24-well culture plates, and incubated at 37°C for 12 to 16 hours in CO 2 Culture in an incubator, add serum-free DMEM medium containing 4 μM agonist WIN 55, 212-2 at 37°C in CO 2 Cultivate in the incubator for 4 to 6 hours, and then detect the luciferase activity with a chemiluminescence detector. The results are shown in Figure 4 .

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Abstract

The invention relates to a novel G protein-coupled receptor (GPCR) cell horizontal screening system as well as a construction method and an application. The screening system is constructed by HEK293 cells or CHO cells, and contains two fusion expression vectors constructed by gene sequences of different groups from the following sequences: the first group: (1) an assembly sequence of a C-terminal of a DnaE gene and a C-terminal of a report gene: DnaE-C-Report-C, (2) an assembly sequence of an N-terminal of the DnaE gene and an N-terminal of the report gene: Report-N-DnaE-N; and the second group: (1) GPCR gene, and (2)Beta-arrestin gene. The system and the method and the application of the invention mainly have the advantages of: high sensitivity and strong specificity when applying the screening system for G protein-coupled receptor (GPCR) cell horizontal screening, simple and fast operation and wide detection range, and can screen agonists and antagonists of any GPCR or GPCR signal transduction approach in any target cell type.

Description

(1) Technical field [0001] The present invention relates to a novel G protein-coupled receptor (GPCR) cell-level screening system and a construction method thereof, and the G protein-coupled receptor screening system for screening agonists or antagonists of GPCR or GPCR signal transduction pathways application in agents. (2) Background technology [0002] G protein-coupled receptors (GPCRs) are the largest receptor family on the cell surface and one of the most diverse protein families. GPCRs share a common secondary structure feature - 7 α-helical transmembrane structures, the N-terminal is outside the cell membrane, and the C-terminal is located inside the cell membrane, so it is also called 7 transmembrane receptors. GPCR is responsible for extracellular signal transmission These chemical signaling molecules include: ions (acting on the parathyroid and renal chemosensitivity Calcium ions of detectors), amino acids (glutamate and aminobutyric acid), monomeric amines (cat...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12N5/10C12N15/62C12N15/85G01N33/68
Inventor 周耐明
Owner ZHEJIANG UNIV
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