Construction And Expression Of Bait Vector For The Recombinant Plasmid SARP1 Gene And Detection Of Their Self-activation In Yeast Two-hybrid Analysis.

Objective: To construct SARP1 ( secreted apoptosis-related protein 1)yeast bait vector for purpose of identifying the interacted proteins and studying the biological functions of the SARP1 gene in the scar tissue.Methods: Using PCR to amplify the target gene from SARP1 gene full-length DNA segment by designing the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I. Selecting pTA2 as the cloning vector. Digesting the vector pGBKT7 and pTA2-SARP1 PCR production with Nde I and Sal I. Ligating the vector and the PCR production to construct pGBKT7-SARP1 recombination. Then pGBKT7-SARP1 target gene segments were transferred into E.coli DH5αcompetent cells, and cultured in LB agar medium which contained kanamycin for selecting positive clones. The integrity and fidelity of SARP1 gene contained in the recombinant plasmids from positive clones were identified by double digesting with Nde I and Sal I endonucleases and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 and screened the positive clone on the plate of SD/-Trp according to Yeast Transformation System 2 manual book (clontech). The positive clones were identified by PCR, and its toxicity and transcriptional activation was tested by the phenotype assay.Results: SARP1 gene sequence contained in recombinant plasmid pGBKT7-SARP1 was verified by enzymes digestion and DNA sequencing analysis. The sequence of inserted SARP1 gene was same as the corresponding sequence found in Gene Bank database (Gene bank ID : AF017986). The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could grow white colonies on SD/-Trp plates and none could survive on SD/-Leu plates.Conclusion: The recombinant yeast pGBKT7-SARP1 bait vector has no biological toxicity and it can not activate the transcription of the reporter gene alone. The recombinant yeast pGBKT7-SARP1 bait vector was constructed successfully, which provides a useful tool for analyzing SARP1 gene's function as well as establishing a molecular platform and advances the functional study of the mechanisms of the scar.