应用酵母双杂交系统研究与血管紧张素-(1-7)相互作用的蛋白

Study on Proteins Interacting with Angiotensin-(1-7) by Yeast Twohybrid System

目的用酵母双杂交系统筛选与血管紧张素-(1-7)相互作用的蛋白,为该7肽在体内如何发挥功能提供线索。方法构建Ang-(1-7)的真核表达载体pBD—Ang-(1—7),转化酵母菌YRG-2,进行毒性和自身非特异激活性检验。与大鼠心肌细胞文库质粒共同转化酵母细胞,在营养缺陷培养基上进行双杂交筛选,选择既能在3重营养缺陷培养基上生长,也能使X—gal变蓝的克隆为阳性,提取靶质粒后进行复交,将真阳性质粒测序,进行生物信息学分析。结果诱饵载体构建成功并转化酵母,对酵母无毒性,无自身激活现象。筛选出的蛋白主要参与细胞代谢和蛋白合成。结论Ang-(1-7)可能影响细胞内某些蛋白合成和细胞代谢,发挥对血管紧张素Ⅱ的结抗作用。酵母双杂交结果为Ang-(1—7)的作用途径的进一步研究提供了分子生物学线索。

Objective To obtain proteins interacting with angiotensin-(1-7). Methods The recombinant bait plasmid pBD Ang- (1-7) was constructed and transformed into yeast strain YRG-2 to test pBD-Ang-(1-7) for non-specific activation. Rat cardiac myocyte cDNA libraries were screened with a two-hybrid system, and colonies which could grow on SD/trp-leu-his medium and had β-gal activity were obtained as true positive target plasmid. The inserts of the target plasmids were analyzed by sequencing and basic local alignment sequence tool (BLAST). Results The recombinant bait vector, pBD-Ang-(1-7) was confirmed by sequencing and was transformed into yeast strain YRG-2. The transformnants showed no autonomous activation and no toxicity on YRG-2. The proteins obtained are mainly involved in the celluar metabolism and proteins sythesis. Conclusion Angiotensin-(1-7) may interfere with cell metabolism and protein synthesis, disturb balance among transcription factors, exert antagonistic action on Angiotensin II. This investigation provides a functional clue at molecular level for the further exploration of the septopeptide.