Construction and Identification of a Bait Vector for the Mature Peptide of Wap65-2 of Large Yellow Croaker in Yeast Two-Hybrid System

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.016

Abstract

Abstract: The mature peptide of Wap65-2 gene（LcWap65-2m）in large yellow croaker（Larimichthys crocea）was used to construct the yeast two-hybrid bait vector, and its self-activation and toxic effect were measured. LcWap65-2m from the liver cDNA of large yellow croaker was amplified, and cloned into the plasmid pGBKT7. After verification by PCR amplification, enzyme digestion and sequencing, the bait vector pGBKT7-LcWap65-2m was transformed into the yeast strain Y187. The self-activation and toxic effect of the recombinant protein were detected in selective nutrition medium, and the expression of the bait protein LcWap65-2m was analyzed by Western blot. LcWap65-2m was successfully amplified and inserted into the plasmid vector pGBKT7. Western blot analysis showed that the bait protein LcWap65-2m was expressed in yeast cells;phenotypic screening tests revealed that the bait protein LcWap65-2m had no self-activation activity and toxic effect. In conclusion, the yeast two-hybrid bait vector pGBKT7-LcWap65-2m was successfully constructed, which laid the foundation for screening the protein interacted with the bait protein Wap65-2 using the yeast two-hybrid system.

Key words: Wap65-2, yeast two-hybrid, expression, self-activation, toxic effect

Qiu Tianxiu, Li Changhong, Chen Jiong. Construction and Identification of a Bait Vector for the Mature Peptide of Wap65-2 of Large Yellow Croaker in Yeast Two-Hybrid System[J]. Biotechnology Bulletin, 2015, 31(8): 108-113.

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