Method for detecting interaction between Rab protein and its effector factor based on fluorescence resonance energy transfer method

A fluorescence resonance energy and effect factor technology, applied in the field of biological sciences, can solve problems such as inability to obtain results, high noise, and low sensitivity, and achieve the effects of simple methods, high resolution of false positives, and high resolution

Inactive Publication Date: 2017-07-07
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the study of using mantGTP as a fluorescent emission group to detect the interaction between Rab protein and effector, the effect is not ideal, because when the effector binds to Rab:mantGTP, its molecular switches I and II change in structure It is not very large, so the interaction between them is often not detected by the direct fluorescence method. At present, mantGTP is often used as the fluorescent emission group, and the fluorescence polarization method is used to detect
The method of fluorescence polarization is based on using vertically polarized light to excite the fluorescent emitting group, and the intensity of the emission spectrum can be detected on the vertical and horizontal polarization planes. This method has a great relationship with the size of the detected molecule. If the effector molecule If it is very small, satisfactory results are often not obtained
In addition, the method of fluorescence polarization is relatively noisy and has low sensitivity, which limits the detection of the interaction between Rab protein and effectors

Method used

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  • Method for detecting interaction between Rab protein and its effector factor based on fluorescence resonance energy transfer method
  • Method for detecting interaction between Rab protein and its effector factor based on fluorescence resonance energy transfer method
  • Method for detecting interaction between Rab protein and its effector factor based on fluorescence resonance energy transfer method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] The AtRabE1d protein in Arabidopsis is a member of the Rab8 subfamily of the small G protein Rab family. It plays an important role in the vesicle transport from the trans-Golgi membrane to the plasma membrane. AvrPto is a member of the Pseudomonas syringae tomato Pathogenic variants are secreted into Arabidopsis thaliana through the III secretion system. In the past two years, AvrPto was screened by yeast two-hybrid method as an effector, which may interact with the GTP-bound AtRabE1d protein (Speth EB, Imboden L , Hauck P, He SY, Plant physiology, 2009, 149, 1824-1837.) as an example.

[0031] (1) construct GFP_pET28a fusion expression vector;

[0032] The pCAMBIA1302 plasmid was used as a template, GFP_forward and GFP_reverse were used as primers, PCR amplification was performed to obtain the target gene, and then the obtained target gene and pET28a vector were double-digested with NcoI / BamHI, and the digested products were collected, and then passed through T4 ligas...

Embodiment 2

[0061] Example 2: AtRabA4b is a member of the Rab family in Arabidopsis thaliana, and recent literature reports have identified a new effector Plant U-BOX13 (PUB13) of it by the method of yeast two-hybrid, and the UND and U of PUB13 are mentioned in the literature -box domain (amino acid sequence 1-370) can only interact with GTP-bound AtRabA4b (Antignani V, Klocko AL, Bak G, Chandrasekaran SD, Dunivin T, Nielsen E, The Plant Cell, 2015, 27, 243-261 .).

[0062] (1) construct GFP_pET28a fusion expression vector;

[0063] The pCAMBIA1302 plasmid was used as a template, GFP_forward and GFP_reverse were used as primers, PCR amplification was performed to obtain the target gene, and then the obtained target gene and pET28a vector were double-digested with NcoI / BamHI, and the digested products were collected, and then passed through T4 ligase at 16°C After connecting overnight, the GFP_pET28a expression vector was successfully constructed.

[0064] The primers used are:

[0065]...

Embodiment 3

[0090] Example 3: Rab8a is a member of the Rab family in human origin, and one of its effectors, inositol 5-phosphatase OCRL1, has been reported in the literature 540-678 Interacts with Rab8a (Hou XM, Hagemann N, Schoebel S, Blankenfeldt W, Goody RS, Erdmann KS, Itzen A, The EMBO Journal, 2011, 30, 1659-1670).

[0091] (1) construct GFP_pET28a fusion expression vector;

[0092] The pCAMBIA1302 plasmid was used as a template, GFP_forward and GFP_reverse were used as primers, PCR amplification was performed to obtain the target gene, and then the obtained target gene and pET28a vector were double-digested with NcoI / BamHI, and the digested products were collected, and then passed through T4 ligase at 16°C After connecting overnight, the GFP_pET28a expression vector was successfully constructed.

[0093] The primers used are:

[0094] GFP_forward-CCATGGGCCATCATCATCATCATCACGTAGATCTGACTAGTAAAGGAGAA;

[0095] GFP_reverse-CGC GGATCC TTTGTATAGTTCATCCATGCCA.

[0096] BamHI

[0097...

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Abstract

The invention belongs to the technical field of bioscience and particularly relates to a method for detecting interaction between Rab protein and its effector factor based on fluorescence resonance energy transfer method. The method comprises: using fluorescent analog mantGTP of natural GTP to replace GDP binding with Rab protein to obtain a donor of fluorescence resonance energy transfer; constructing a vector for fusion expression of the effector factor of the Rab protein and green fluorescence protein GFP, and using the expression-purified fusion protein as a receptor of fluorescence resonance energy transfer. It is possible to detect, directly in vitro, the interaction between the Rab protein bound in mantGTP form and the effector factor fusion-expressed with the green fluorescence protein GFP by using a fluorescence spectrophotometer based on the fluorescence resonance energy transfer method. The novel method for sensitively and quickly detecting interaction between Rab protein and its effector factor, and the protein interaction system is completed.

Description

technical field [0001] The invention belongs to the technical field of biological sciences, and specifically relates to a method for detecting the interaction between Rab protein and its effector based on the fluorescence resonance energy transfer method. Background technique [0002] Rab protein is the largest subfamily in the small molecule GTP-binding family, and it is a monomeric GTP-binding protein. It cycles between the GTP-bound active form and the GDP-bound inactive form. Different forms of Rab protein will change in structure. The region where this conformation changes is called the molecular switch region, including molecular switch I (Switch I) and Molecular Switch II (Switch II). Molecular switches I and II are the key sites for the binding of Rab protein and its interaction factors. The conversion of Rab protein between these two nucleotide binding forms can be catalyzed by its upstream regulatory proteins, such as guanine nucleotide exchange factor (guanine n...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 侯晓敏高怡董春海
Owner QINGDAO AGRI UNIV
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