Fluorescent protein iRFP-based dimolecular fluorescent fragment complementary system, and applications

A fluorescent protein and bimolecular technology, applied in the field of bimolecular fluorescent fragment complementary systems, can solve the problems of inability to intuitively display the inhibition effect, lack of screening and verification methods, etc., and achieve the effect of simple inhibition effect.

Active Publication Date: 2016-06-01
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of these methods are based on in vitro studies and cannot directly demonstrate their inhibitory effects, and the screened lead compounds need to be further verified at the cellular level or in vivo.
Current screening and validation methods are lacking

Method used

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  • Fluorescent protein iRFP-based dimolecular fluorescent fragment complementary system, and applications
  • Fluorescent protein iRFP-based dimolecular fluorescent fragment complementary system, and applications
  • Fluorescent protein iRFP-based dimolecular fluorescent fragment complementary system, and applications

Examples

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Embodiment 1

[0044] A construction of a bimolecular fluorescent fragment complementation system based on the fluorescent protein iRFP97-98 site, the construction process includes the following steps:

[0045] (1) PCR amplification (upstream Primer: 5'-GAAGATCTATGGCTGAAGGATCCGTC-3', downstream primer: 5'-AAGGTACCCATCGTGAAGCCGACAGTG-3') to obtain the gene sequence of the nitrogen-terminal fragment iRN97 of the fluorescent protein iRFP from bacteria, and use the double restriction sites BglII and KpnI to convert the iRN97 gene Inserted into the multiple cloning site of the pEGFP-C1 (purchased from Clontech) vector to construct the vector pEGFP-iRN97;

[0046] (2), from the plasmid pcDNA3.1-iRFP by PCR amplification (upstream primer: 5'-GAAGATCTATGATGCGAAAGGACG-3', downstream primer: 5'-CCCAAGCTTCCTCTTCCATCACGCCGATCTGCCAGG-3') to obtain the carbon-terminal fragment of the fluorescent protein iRFP derived from bacteria For the gene sequence of iRC98, the iRC98 gene was inserted into the multip...

Embodiment 2

[0053] A bimolecular fluorescent fragment complementation system based on fluorescent protein iRFP, the construction process includes the following steps:

[0054] 1) Obtain the gene sequence of the nitrogen-terminal fragment iRN97 of the bacterial fluorescent protein iRFP from the plasmid pEGFP-iRN97 by PCR amplification (upstream primer: 5'-GGGATATCGAGCCACCCCCTCCTATGGCTGAAGGATCCGTC-3', downstream primer: 5'-CCGCTCGAGTTACATCGTGAAGCCGACAG-3') (shown in SEQIDNO.2), utilize the double restriction site EcoRV and XhoI, iRN97 gene is inserted on the multiple cloning site of pcDNA3.1 vector, namely plasmid piRN97( Figure 1-A );

[0055] 2) Obtain the gene sequence of the carbon-terminal fragment iRC98 of the bacterial fluorescent protein iRFP from the plasmid pEGFP-iRC98 by PCR amplification (upstream primer: 5'-CGGCTAGCGCCACCATGCGAAAGGACGCAGGCTTCATC-3', downstream primer: 5'-CCCAAGCTTCCTCTTCCATCACGCCGATCTGCC-3') (shown in SEQIDNO.3), utilize double-digestion site NheI and HindIII,...

Embodiment 3

[0057] The application of a fluorescent protein iRFP-based bimolecular fluorescent fragment complementation system in the study of protein interactions, the application process is as follows:

[0058] 1) The synthetic bJun gene sequence (synthesized by Shanghai Sangong, refer to the literature HuCDetal.MolCell.2002; 9:789-98.), using the double restriction sites NheI and HindIII, inserting the bJun gene into the multiple clone of the piRN97 vector site, construct the vector pbJun-iRN97;

[0059] 2) The gene sequence of the synthetic bFos (synthesized by Shanghai Sangong, refer to the literature HuCDetal.MolCell.2002; 9:789-98.), using the double restriction site KpnI and NotI, insert the bFos gene into the multiple clone of the plasmid piRC98 site, construct the vector piRC98-bFos;

[0060] 3) The control group is a plasmid constructed by using the upstream primer 5'-GGGGTACCGAGCCACCCCCTCCTATGGGTCGTGCGCAGTC-3' and the downstream primer 5'-ATTTGCGGCCGCTTAACCCAGGTCGTTCGGGATTTTG...

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Abstract

The invention discloses a fluorescent protein iRFP-based dimolecular fluorescent fragment complementary system, and applications. According to the fluorescent protein iRFP-based dimolecular fluorescent fragment complementary system, phytochrome fluorescent protein iRFP is taken as a template, and is divided into a non-fluorescent nitrogen terminal fragment iRN97 and a non-fluorescent carbon terminal fragment iRC98 at the amino acid 97-98 sites; when fusion expression of the two fragments with protein pairs with interactions is realized, the two non-fluorescent fragments are drawn to be close to generate iRFP fluorescence; fusion expression of iRN97 with human immunodeficiency virus integrase IN, and fusion expression of iRC98 with cell protein P75 are realized respectively, the interaction between IN and p75 is studied in living cells via fluorescent complementation of iRN97 with iRC98; when a drug is capable of inhibiting the protein-protein interaction, it is impossible for iRN97 and iRC98 to be drawn to be close, so that fluorescence recovery is inhibited. The fluorescent protein iRFP-based dimolecular fluorescent fragment complementation system is a simple, effective, and convenient evaluation system of drugs used for inhibiting protein interactions.

Description

technical field [0001] The invention relates to the technical field of drug evaluation for inhibiting protein interaction, and more specifically relates to a fluorescent protein iRFP-based bimolecular fluorescent fragment complementation system and its application. Background technique [0002] Fluorescent Fragment Complementation System is a kind of fragment complementation system using fluorescent protein as material. The basic principle is to split the fluorescent protein into nitrogen-terminal and carbon-terminal fragments at appropriate sites, and if the two fragments are not under the action of interacting proteins, they cannot spontaneously complement each other to restore fluorescence. If a pair of interacting proteins are respectively fused to the nitrogen-terminal and carbon-terminal fragments of the split fluorescent protein, under the action of this pair of interacting proteins, the nitrogen-terminal fragment and the carbon-terminal fragment of the split fluoresc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66G01N21/64
Inventor 崔宗强张先恩陈明海张治平李炜
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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