Protein fluorescence localization detecting system and method for fast detecting protein interaction and application thereof

A protein interaction and positioning detection technology, applied in the fields of biotechnology and genetic engineering, can solve the problems of prone to false positives, many false positives, and can not be guaranteed, and achieve the effect of eliminating false positives

Inactive Publication Date: 2016-04-20
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, different methods have different advantages and disadvantages. For example, the co-immunoprecipitation method is complicated to operate and cannot guarantee that the precipitated complex is a direct interaction protein; the yeast two-hybrid method can only verify the interaction in the nucleus, and is prone to false positives ; Bimolecular fluorescence complementation and fluorescence resonance energy transfer methods are relatively simple and convenient to operate, but there are many problems of false positives

Method used

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  • Protein fluorescence localization detecting system and method for fast detecting protein interaction and application thereof
  • Protein fluorescence localization detecting system and method for fast detecting protein interaction and application thereof
  • Protein fluorescence localization detecting system and method for fast detecting protein interaction and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1. Protein fluorescence localization detection carrier construction

[0036] Log in to the NCBI database and download the currently known nucleotide sequences expressing DsRed and EGFP proteins, the sequences of which are shown in SEQIDNo.1 and SEQIDNo.2 respectively, according to the multiple cloning site of the pIZ-V5 / His (Invitrogen Company) vector Design corresponding primers with nuclear localization signal sequence and without nuclear localization signal sequence, and then send them to BGI for synthesis. The primer sequences are as follows:

[0037] EGFP-F:5'-cccaagcttatggtgagcaagggcgaggagct-3', (SEQ ID No.10);

[0038] EGFP-R:5'-ggggtacccttgtacagctcgtccatgcc-3', (SEQ ID No.11);

[0039] NSL-EGFP-F:5'-cccaagcttccgaagaagaagcgaaaggtggaagacccgggaacgatggtgagcaagggcgaggagct-3', (SEQ ID No. 12);

[0040] DsRed-F:5'-cccaagcttatggcctcctccgagaacgtcat-3', (SEQ ID No.13);

[0041] DsRed-R: 5'-ggggtacccaggaacaggtggtggcgg-3', (SEQ ID No. 14);

[0042] NSL-DsRed-F: ...

Embodiment 2

[0046] Example 2, Verification of Protein Interaction Identification by Protein Fluorescence Localization Detection System

[0047] The protein fluorescent localization carrier constructed in Example 1 can be localized to the corresponding position. In order to verify that this system can observe whether there is an interaction between the two proteins through the change of fluorescent localization, first analyze that LEF-11 has a nuclear localization signal, and select pIZ- EGFP and pIZ-DsRed were used as target vectors, and a full-length fusion vector pIZ-DsRed-LEF11 / pIZ-EGFP-LEF11 of LEF-11 (sequence shown in SEQIDNo.4) full-length fusion of DsRed and EGFP was constructed, and a mutant LEF-11 was constructed at the same time The key amino acid mutant sequence of the nuclear localization signal at position 100 is shown in SEQ ID No.5; the vector pIZ-LEF11(K100L)-DsRed fused with DsRed, and the control vector is pIZ-EGFP. The vector construction method is the same as that in ...

Embodiment 3

[0051] Example 3. Application of protein fluorescence localization detection system in the study of LEF-11 protein interaction with nuclear localization signal

[0052] In order to study the practicability of the protein fluorescence localization detection system described in Example 1 in identifying protein interactions, the N-terminus of LEF-11 containing only one oligomerization domain (amino acid 42-61 region) was truncated and mutated, And a fluorescent protein fusion expression vector was constructed, see for details Image 6 Schematic diagram of fluorescent protein construction of LEF-11 N-terminal truncation mutant vector. The specific construction method of the vector is the same as that in Example 1, and all the vectors have been verified by enzyme digestion and sequencing.

[0053] The constructed fluorescent protein fusion expression vector pIZ-DsRed-LEF11(aa2-61), pIZ-DsRed-LEF11(aa12-61), pIZ-DsRed-LEF11(aa22-61), pIZ-DsRed-LEF11(aa32-61 ), pIZ-DsRed-LEF11(aa42...

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Abstract

The invention discloses a protein fluorescence localization detecting system and a method for fast detecting protein interaction and application thereof. The protein fluorescence localization detecting system is composed of fusion protein composed of fluorescent protein A and protein A to be detected, fusion protein composed of fluorescent protein A, nuclear localization signals and protein A to be detected, fusion protein composed of fluorescent protein B and protein B to be detected, and fusion protein composed of fluorescent protein B, nuclear localization signals and protein B to be detected, wherein the fluorescent protein A and the fluorescent protein B are different in fluorescence color, and the protein A to be detected and the protein B to be detected are two kinds of protein to be detected whether interaction exists or not. Whether the two kinds of protein interact or not can be detected in a cell through the method, and the false positive phenomenon of fluorescence dimolecular complementation technology and fluorescence resonance energy transfer can be eliminated.

Description

technical field [0001] The invention belongs to the technical fields of biotechnology and genetic engineering, and relates to a method for rapidly detecting protein interaction through protein fluorescence positioning. Background technique [0002] Protein interaction network is the main manifestation of biological information regulation and a key factor in determining cell life activities. Interpreting the interaction between proteins is a crucial part of understanding the biological activities of cells, the signal transduction mechanism of proteins and biological metabolism and other life processes. It can help determine the role of proteins in the whole life process role provides decisive clues. [0003] At present, the most commonly used methods for protein interaction research are co-immunoprecipitation (Co-immunoprecipitation, Co-IP), yeast two-hybrid system (YeastTwo-HybridSystem), fluorescence resonance energy transfer (Fluorescent Resonant Energy Transfer, FRET), b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62G01N21/64
CPCC07K14/005C07K14/43586C07K2319/09C07K2319/60C12N2710/14122G01N21/6486
Inventor 潘敏慧鲁成董战旗陈婷婷胡楠董非凡
Owner SOUTHWEST UNIVERSITY
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