Recombinant vector for high-throughput yeast two-hybrid technology and method for large-scale screening of interacting proteins

Abstract

The invention discloses a related recombinant vector used for yeast two-hybrid and a yeast two-hybrid method for large-scale screening interacting protein using a high-flux sequencing technology. The method depends on a peculiar integrative function of an integrase Integrase phiC31(En), and by conducting transformation on a traditional yeast two-hybrid vector, the vector is capable of conducting organic combination with the high-flux sequencing technology, and thus that hundreds of bait protein can simultaneously conduct screening work on Prey protein library can be realized. The method comprises the steps of inserting the integrase and a specially recognized nucleotide sequence into a vector of a Matchmaker GAL4 system, making the recombinant vector enter into the same yeast cell through the yeast two-hybrid method, using the integrase to play a role in conducting specific orientational rearrangement on the specially recognized nucleotide sequence and a nucleotide sequence closely adjacent to the specially recognized nucleotide sequence so as to make two recombinant vectors combine into a combined vector, at this time the Prey protein and the bait protein are orientationally connected into one new section of sequence on the combined vector. Through amplification and high high-flux sequencing, the recombinant vector can conduct economic and efficient screening on interacting proteins and construct a comprehensive network atlas for interactions between the proteins.

Description

technical field [0001] The invention relates to a yeast two-hybrid screening method, in particular to a yeast two-hybrid related carrier and a yeast two-hybrid method for large-scale screening of interacting proteins using high-throughput sequencing technology. Background technique [0002] Yeast two-hybrid technology, as the main method and means to study large-scale protein interaction, has been greatly developed and more and more widely used. [0003] Yeast two-hybrid system, as the most commonly used technical means to study the interaction between proteins, was first established by Fields and Song in the study of eukaryotic gene transcription regulation. The principle is: bait protein (bait) and BD (DNA Binding Domain) , the prey protein (pery) and AD (DNA Activation Domain) form fusion proteins respectively, and then the two fusion proteins enter the same yeast cell through yeast hybridization. If there is an interaction between the bait protein and the pery protein, B...

AI Technical Summary

Problems solved by technology

Complex operation, heavy workload and time-consuming

When multiple or even hundreds of Bait proteins are to be screened for interacting proteins, each bait Bait protein and the prey Prey protein library can only be screened separately, and all bait Bait proteins and prey Prey protein libraries cannot be screened simultaneously , the waste of human resources, etc. caused by it is unbearable for ordinary laboratories, so that it is impossible to quickly establish an information-rich protein interaction network

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