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Method for detecting interaction of proteins

A protein interaction and detection method technology, applied in biological testing, material inspection products, fluorescence/phosphorescence, etc., can solve the problems of long experimental period, low sensitivity, cumbersome operation, etc., and achieve high-resolution detection and research effects

Inactive Publication Date: 2012-07-18
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a sensitive, fast, high-resolution protein interaction detection method that detects at the level of a single bacterium in view of the defects of the existing protein interaction detection and analysis methods, such as cumbersome operation, low sensitivity, and long experiment cycle

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0029] The construction of embodiment 1 bacterial two-hybrid system

[0030] Using the Escherichia coli K12 genome as a template, primers for pal and tolB genes were designed and synthesized in Yushengong, and pal and tolB genes were obtained by PCR. They were respectively inserted into the EcorI and XhoI restriction sites of bacterial two-hybrid vectors pUT18 and pKT25 to obtain pUT18-pal and pKT25-tolB. The two vectors carrying pal and tolB were co-transformed into the reporter bacterium E.coli BTH101 to complete the construction of the bacterial two-hybrid system. The successful establishment of the Pal-TolB bacterial two-hybrid system was confirmed by routine blue-white screening and colorimetric quantitative methods, and was verified by traditional biological experiments such as Western Blot, co-immunoprecipitation, and fluorescence microscopy.

Embodiment 2

[0031] The cultivation of embodiment 2 two-hybrid bacteria and the expression of interacting protein

[0032] Pick a single colony of E.coli BTH101+pUT18-pal+pKT25-tolB in 2mL LB medium, add the inducer IPTG to a final concentration of 0.5mM, and culture at 30°C and 250rpm for 18h to induce the expression of Pal and TolB. The interaction of the two proteins promotes the transcription and expression of the reporter gene product β-gal. At the same time, BTH101pKT25+pUT18; BTH101pKT25-TolB+pUT18; BTH101pKT25-+pUT18-Pal were set as negative controls.

Embodiment 3

[0033] Example 3 Determination of β-gal enzyme activity by UV-visible spectrophotometry to determine protein interaction

[0034] The OD600 values ​​of the four bacterial solutions in Example 2 were measured with a spectrophotometer, and the OD600 of the four bacterial solutions were basically consistent by adjusting the amount of M63 medium added. Using the Miller method, ONPG was used as a substrate, and the β-gal enzyme activity of the four bacteria was measured on a UV-visible spectrophotometer (set DU800, kinetic / time, analytical 420nm, Scan time 20min, interval 2min), so as to determine the protein interaction situation.

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Abstract

The invention discloses a method for detecting interaction of proteins and relates to a detection method of proteins. The detection method comprises the following steps of: respectively cloning two interacting proteins to be detected on a bacterial two-hybrid vector and co-transforming to report bacteria; starting transcriptional expression of the report genes because of the interaction of the two target proteins, and generating special enzymes; dyeing the report bacteria co-transformed with the two target proteins by using a fluorogenic substrate, hydrolyzing the fluorogenic substrate by using the report product, that is, enzyme, to release a fluorescein, and detecting the interaction of the two target proteins in a single-bacterium level by quantitatively analyzing fluorescence intensity of the single bacterium by using a flow cytometer. The quantitative detection and correlation analysis of the protein-protein interaction in the single-bacterium level are important for accurate analysis of protein functions, certainty of signal transduction paths and discovery of new drug targets.

Description

technical field [0001] The invention relates to a protein detection method, in particular to a method for rapidly detecting protein-protein interaction at a single bacterium level by combining flow cytometry detection technology and a bacterial two-hybrid system. Background technique [0002] Proteins are the main executors of various physiological functions in organisms, and the interaction between proteins constitutes an important part of the cell biochemical reaction network, which can not only reveal the functions of proteins at the molecular level, but also provide us with a better understanding of Life processes, disease mechanisms, and new drug development provide a good foundation. Protein interaction research methods are divided into two categories: in vitro and in vivo detection, in which in vitro detection mainly includes immunoprecipitation, surface plasmon resonance analysis, phage display, protein chip, etc. ([1] H. Guan, E. Kiss-Toth, Advanced Technologies fo...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/64
Inventor 颜晓梅吴丽娜汪旭
Owner XIAMEN UNIV
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