Experimental method for screening polypeptide library through yeast two-hybridization

Abstract

The invention relates to the technical field of fermentation yeast screening genes, and discloses an experimental method of a yeast two-hybrid screening polypeptide library, which comprises the following steps: preparation of materials: bait clone pGBKT7-gA9-nosc-221, a bait carrier pGBKT7, library yeast plasmid pGADT7-polypeptide cDNA, a Clontech yeast two-hybrid system and a culture medium are prepared; the culture medium is prepared from an escherichia coli culture medium, a yeast complete culture medium, a yeast defect type screening culture medium and other stock solutions. The method specifically comprises the following steps: transforming bait plasmids into recipient bacteria AH109, carrying out self-activation detection on the bait plasmids, carrying out library screening, carrying out yeast positive clone identification and sequencing comparison, carrying out yeast positive clone rotation verification, screening a yeast two-hybrid polypeptide library by taking a gA9-nosc-221 gene constructed on a pGBKT7 vector as bait, and carrying out multiple report gene detection, DNA sequencing and BLAST comparative analysis on the positive clone, so as to obtain the yeast two-hybrid polypeptide library. According to the method, the polypeptide interacting with the pGBKT7-gA9-nosc-221 is determined, the method for determining the polypeptide protein is more efficient, and the research efficiency is greatly improved.

Description

technical field [0001] The invention relates to the technical field of fermented yeast screening genes, in particular to an experimental method for yeast two-hybrid screening of polypeptide libraries. Background technique [0002] Gene (genetic factor) is the entire nucleotide sequence required to produce a polypeptide chain or functional RNA. Gene supports the basic structure and performance of life, and stores all the processes of life's race, blood type, gestation, growth, and apoptosis. The interdependence of information, environment and heredity deduces important physiological processes such as reproduction of life, cell division and protein synthesis. The internal factors that determine the health of life, therefore, genes have dual attributes: material and informative, DNA segments with genetic information are called genes, and other DNA sequences, some directly play a role in their own structure, and some are involved in regulating genetic information The performanc...

AI Technical Summary

Problems solved by technology

[0004] Aiming at the deficiencies of the prior art, the present invention provides an experimental method for yeast two-hybrid screening of polypeptide libraries, which has the ability to use the gA9-nosc-221 gene constructed on the pGBKT7 vector as bait to screen yeast two-hybrid polypeptide libraries. Cloned multiple reporter gene detection, DNA sequencing and BLAST comparison analysis, to determine the advantages of peptides interacting with pGBKT7-gA9-nosc-221, to solve the current gene research efficiency is slow, the research method is not simple enough