Library versus library yeast two-hybrid massive interaction protein screening method

A yeast two-hybrid, protein technology, applied in the field of molecular biology, can solve problems such as incompatibility, and achieve the effects of improving efficiency, low hardware requirements, and simple operation

Inactive Publication Date: 2015-04-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In summary, the existing high-throughput yeast two-hybrid technology is not suitable for large-scale protein-protein interaction screening in general laboratories. New high-throughput yeast two-hybrid method with low hardware requirements

Method used

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  • Library versus library yeast two-hybrid massive interaction protein screening method
  • Library versus library yeast two-hybrid massive interaction protein screening method
  • Library versus library yeast two-hybrid massive interaction protein screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: A library-to-library yeast two-hybrid method for large-scale screening of interacting proteins: Screening of proteins interacting between the plant pathogen Rhizoctonia solani AG Ⅰ-IA and the host corn, the specific steps are as follows:

[0046] (1) Synthesize the "bait" cDNA-Rhizoctonia solani AG Ⅰ-IA cDNA and the "prey" cDNA-corn cDNA respectively:

[0047] a. Trizol reagent from Invitrogen Company extracted the total RNA of Rhizoctonia solani AG Ⅰ-IA and corn leaves inoculated and infected with Rhizoctonia solani AG Ⅰ-IA, and purified the mRNA. -IA mRNA 323.2 ng / μl, maize mRNA 309.6 ng / μl.

[0048] b. Add the following reagents to the RNAase-free microcentrifuge tube in order

[0049] mRNA

1.5μl

GBK-CDS Ⅲ (Rhizoctonia solani AG Ⅰ-IA) / CDS Ⅲ (corn)

1μl

RNAase-free sterile water

1.5μl

total capacity

4μl

[0050] Mix well and centrifuge to the bottom of the tube.

[0051] Primer GBK-CDS Ⅲ was synthesize...

Embodiment 2

[0103] Example 2 A library-to-library yeast two-hybrid method for large-scale screening of interacting proteins: screening of interacting proteins between plant pathogenic bacteria powdery mildew of wheat and host wheat, the specific steps are as follows:

[0104] (1) Separately synthesize "bait" cDNA-wheat powdery mildew cDNA and "prey" cDNA-wheat cDNA:

[0105] a. Trizol reagent from Invitrogen Company extracted the total RNA of wheat powdery mildew bacteria, wheat leaves inoculated and infected with wheat powdery mildew bacteria, and purified to obtain mRNA.

[0106] b. Add the following reagents to the RNAase-free microcentrifuge tube in order

[0107] mRNA

1.0μl

GBK-CDS Ⅲ (wheat powdery mildew) / CDS Ⅲ (wheat)

1.0μl

RNAase-free sterile water

2.0μl

total capacity

4.0μl

[0108] Mix well and centrifuge to the bottom of the tube.

[0109] c. Incubate at 72°C for 2 minutes, cool on ice for 2 minutes, centrifuge to the bott...

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Abstract

The invention discloses a library versus library yeast two-hybrid massive interaction protein screening method. The method takes a genome-grade cDNA as the bait to screen the genome-grade cDNA library. In the application process of the method, the cDNA library is subjected to a homogenization treatment so as to reduce the masking effect of the high-abundance interaction proteins to the low-abundance interaction proteins, and thus the screening efficiency is improved; and moreover the characteristic of yeast URA3 gene is utilized so as to automatically remove the sequences, which have the self-activating function, in bait cDNA. The invention establish a library versus library yeast two-hybrid massive interaction protein screening method, which has the advantages of high feasibility, low cost, low requirements on experiment conditions, and low workload; and successfully applies the method to a screening of pathogen-host plant interaction proteins.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a library-to-library yeast two-hybrid large-scale screening method for interacting proteins. This method is different from the previous yeast two-hybrid method for high-throughput screening of protein-protein interactions. The present invention uses a library-to-library screening method, which can be conveniently and simply used in molecular biology-related research on the basis of overcoming the shortcomings of the former. Research on protein-protein interactions between different species and between different species in the field. Background technique [0002] Protein is the executor of gene function, and its function is not isolated, but is completed by interacting with other molecules in a criss-cross network. And protein-protein interaction is one of the important forms. At present, there are two main strategies for the construction of protein interaction netwo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B20/00C40B50/06
Inventor 史军伟张婷婷董五辈
Owner HUAZHONG AGRI UNIV
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