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Method for screening interacting protein based on bimolecular fluorescence complementation technique

A technology for green fluorescent protein and protein, applied in the field of screening interacting proteins, can solve the problems of high cost, heavy workload and high fluorescence background value

Active Publication Date: 2017-08-11
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-throughput protein interaction screening using BiFC technology has been reported in mammalian cells and yeast, but there are problems of large workload, high cost and high fluorescence background value respectively.

Method used

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  • Method for screening interacting protein based on bimolecular fluorescence complementation technique
  • Method for screening interacting protein based on bimolecular fluorescence complementation technique
  • Method for screening interacting protein based on bimolecular fluorescence complementation technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1, the establishment of the BiFC system based on YGFP

[0065] 1. Transformation of yeast expression vectors pPC86 and pDBLeu

[0066] 1. Transform yeast expression vector pPC86 and construct pPC86-pADH-linker

[0067] (1) Restriction endonucleases KpnI and SpeI were used to double-digest the yeast expression vector pPC86, remove the ADH promoter-NLS-GAL4AD sequence, and retain a vector backbone fragment with a size of about 5209bp.

[0068] (2) Using pPC86 as a template, using primers pPC86-pADH-F and pPC86-linker-R to carry out PCR amplification, the ADH promoter-linker sequence was amplified, and it was treated with KpnI and SpeI double enzyme digestion and then connected into the step ( 1) The backbone fragment of the pPC86 vector that has been digested with KpnI and SpeI to form the pPC86-pADH-linker vector.

[0069] Upstream primer pPC86-pADH-F:

[0070] 5'-AAAGGTACCATCCGGGATCGAAGAAATG-3';

[0071] Downstream primer pPC86-linker-R:

[0072] 5'-CTAG...

Embodiment 2

[0089] Example 2, using the BiFC system established in Example 1 to verify the interaction between bJun protein and bFos protein

[0090] It is known that bJun protein and bFos protein are a pair of interacting proteins (see "Hu, C.D., Chinenov, Y. & Kerppola, T.K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Molecular cell 9,789-798( 2002).”, the present embodiment selects “bJun protein and bFos protein” that interact with each other as the positive test group, “bJun protein and the ΔbFos protein that has lost the interaction domain with bJun protein” (published “bFos protein interacts with The reference of bJun protein interaction domain" is "Hu, C.D., Chinenov, Y.&Kerppola, T.K.Visualization of interactions among bZIP and Refamily proteins in living cells using bimolecular fluorescencecomplementation. Molecular cell 9,789-798(2002).") as In the negative experiment group, the BiFC system esta...

Embodiment 3

[0145] Example 3, application of the BiFC system established in Example 1 to screen for p53-interacting proteins

[0146] After establishing the YGFP-based BiFC system in yeast, this example uses p53 protein as a bait to screen its interacting proteins from a humanized cDNA library.

[0147] 1. Construction of pDBLEu-YC157-p53 and pDBLEu-p53-YC157 vectors

[0148] 1. Construction of pDBLeu-YC157-p53 vector

[0149] Using the coding gene of p53 protein shown in Sequence 9 in the Sequence Listing as a template, primers were used to perform PCR amplification on p53-F1 and p53-R1 to obtain amplified products with restriction sites XbaI and NotI at both ends.

[0150] p53-F1:5'-ACC TCTAGA ATGGATGATTTGATGCTGTCCC-3';

[0151] p53-R1: 5'-ATTT GCGGCCGC GTCTGAGTCAGGCCCTTCTGT-3'.

[0152] The amplified product was double-digested with restriction endonucleases XbaI and NotI, and after gel recovery, it was ligated with the pDBLeu-YC157 vector (see Example 2 for the specific constru...

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Abstract

The invention discloses a method for screening interacting protein based on the bimolecular fluorescence complementation (BiFC) technique. The method comprises the steps of 1, dividing YGFP into an N end (sequence 1, 1-157) and a C end (sequence 1, 158-238); connecting a coding gene of target protein A to the 3' terminal of an N-end coding gene through a connecting sequence (connecting peptide as shown in coding sequence 2), so as to form a fusion gene fragment A; connecting a coding gene of target protein B to the 5' terminal or 3' terminal of a C-end coding gene through the connecting sequence, so as to form a fusion gene fragment B; and 2, importing the fusion gene fragments A and B into receptor yeast cells, conducting culture to obtain transgenic yeast cells, and detecting whether the transgenic yeast cells generate green fluorescence, wherein an interaction or candidate interaction relation exists between the target protein A and the target protein B if yes, and no interaction or candidate interaction relation exists between the target protein A and the target protein B if not. The BiFC technique has high sensitivity and specificity, and is suitable for high-throughput protein-protein interaction screening in yeast.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for screening interacting proteins based on bimolecular fluorescence complementary technology. Background technique [0002] Proteins constitute the basis of life activities, and the interaction between proteins exists in almost all life activity phenomena. They form complex interaction networks to jointly regulate various life activities. Therefore, a large-scale protein interaction network linkage map The mapping of the protein will help reveal the interaction between proteins, and then clarify the biological function of the protein, so that we can understand the nature of life phenomena at the molecular level. It is estimated that there are 130,000 pairs of interactions in human proteins, but the existing interaction network only reveals a small part of them, so there are still a large number of interacting proteins to be further excavated. [0003] At present, large-scale...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81
CPCC12N15/81C12N2800/102
Inventor 王建贺福初商立民原艳芝
Owner ACADEMY OF MILITARY MEDICAL SCI
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