酵母双杂交技术筛选杜氏盐藻cDNA文库中与S-腺苷高半胱氨酸水解酶相互作用的 蛋白
Screening of proteins that interact with S？？adenosyhomocystine hydrolase by yeast two？？hybrid assay in Dunaliella salina cDNA library

目的：应用酵母双杂交技术筛选杜氏盐藻cDNA文库中能与S？蚕佘崭甙腚装彼崴？解酶(dsSAHH)相互作用的蛋白。方法：将构建好的pGBKT7？？dsSAHH诱饵载体转化至酵母细胞Y187，与转化有杜氏盐藻cDNA文库的酵母细胞AH109杂交，从杜氏盐藻cDNA文库中筛选出能与dsSAHH相互作用的蛋白质。扩增所得基因片段全长，并在原核内表达该融合蛋白。随后采用Far？？western blot、His pull？？down及免疫共沉淀方法验证dsSAHH与目的蛋白在体内、外的相互作用。结果：以dsSAHH为诱饵筛选出3个阳性克隆，扩增得到dsRACK1、dsMetAP1和dseEF1α 3个新基因。His pull？？down和免疫共沉淀体内外实验证实dsRACK1能与dsSAHH相互作用。结论：dsRACK1是dsSAHH的相互作用蛋白。
Aim: To isolate and identify the proteins that interact with S？？adenosyhomocystine hydrolase(dsSAHH) in Dunaliella salina(D.salina) cDNA library. Methods: The constructed bait vector pGBKT7？？dsSAHH was transformed to yeast cell Y187 and then the Y187 cells hybridized with yeast AH109 cells which contained the cDNA library of D.salina. The proteins that interacted with dsSAHH of D.salina were screened and identified using yeast two？？hybrid technology. Then, the full length of screened gene was cloned and the fusion proteins of aimed gene were expressed in E.coli. Finally, the interaction was confirmed by Far？？western blot, His pull？？down, and co？？immunoprecipitation in vitro and in vivo. Results: Three new genes, including dsRACK1, dsMetAP1, and dseEF1α, were screened. Out of them, dsRACK1 interacted with dsSAHH，which was confirmed by Far？？western bolt and His pull？？down. Conclusion: dsSAHH could interact with dsRACK1